CN103354836B - Novel Wine brewing yeast strain - Google Patents
Novel Wine brewing yeast strain Download PDFInfo
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- CN103354836B CN103354836B CN201180054540.2A CN201180054540A CN103354836B CN 103354836 B CN103354836 B CN 103354836B CN 201180054540 A CN201180054540 A CN 201180054540A CN 103354836 B CN103354836 B CN 103354836B
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- bacterial strain
- wood sugar
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- wine brewing
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- 238000011160 research Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 239000007320 rich medium Substances 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
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- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- COBXDAOIDYGHGK-UHFFFAOYSA-N syringaldehyde Natural products COC1=CC=C(C=O)C(OC)=C1O COBXDAOIDYGHGK-UHFFFAOYSA-N 0.000 description 1
- YIBXWXOYFGZLRU-UHFFFAOYSA-N syringic aldehyde Natural products CC12CCC(C3(CCC(=O)C(C)(C)C3CC=3)C)C=3C1(C)CCC2C1COC(C)(C)C(O)C(O)C1 YIBXWXOYFGZLRU-UHFFFAOYSA-N 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 230000004127 xylose metabolism Effects 0.000 description 1
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Abstract
Describe a kind of method for generation of Wine brewing yeast strain, this Wine brewing yeast strain has the gene of encodes xylose reductase, xylitol dehydrogenase and the xylulokinase be introduced into and has the inhibitor tolerance of the ethanol production of improvement, the xylose rate of improvement, the Xylitol output of reduction and improvement.The method comprises in a continuous mode, substantially the substratum of wood sugar as carbon source is only comprised with a kind of, under 25 ° of C-38 ° of C, the preferably temperature of 30 ° of C-35 ° of C and the air-flow of 0.040vvm-0.055vvm, cultivate a kind of Wine brewing yeast strain, increase thinning ratio and maintain constant cell level, described cell levels is determined within the scope of 1.5-3.0 by optical density (OD) or equivalent analysis means, and in these cells, adds at least one inhibitor and little by little increase the add-on of described inhibitor.In addition, the multiple Wine brewing yeast strain that method according to the present invention obtains is described through.
Description
the technology of the present invention field
The present invention relates to a kind of method for generation of yeast saccharomyces cerevisiae (Saccharomycescerevisiae) bacterial strain, this Wine brewing yeast strain has the gene of encodes xylose reductase, xylitol dehydrogenase and the xylulokinase be introduced into, and has the inhibitor tolerance of the ethanol production of improvement, the xylose rate of improvement, the Xylitol output of reduction and improvement.The present invention relates in addition by the obtainable multiple Wine brewing yeast strain of method according to the present invention.
Background technology
About using oil as the environmental problem of automobile fuel and also having current oil well in the future depleted danger to be caused the further investigation of the replacement scheme about use oil.It finds that ethanol is a kind of good surrogate of oil, because can replace oil use and can not cause the great change of oil engine to a great extent.Can very little adjustment done to engine or even use when not doing any adjustment to engine ethanol to replace some fuel now.
Saccharomycodes (Saccharomyces) bacterial strain is widely used in for brewageing, distill, toast and in other different industry of applying.In view of sugar (as dextrose plus saccharose) is converted into biomass and is the ability of ethanol by these sugar-fermentings by yeast saccharomyces cerevisiae, yeast saccharomyces cerevisiae is one of most popular microorganism in industrial application.Quite promptly sugar changed into the ability of ethanol in view of Wine brewing yeast strain and have better tolerance than to fermentation inhibitor and ethanol due to yeast saccharomyces cerevisiae and bacterium and other Yeast Phases, Wine brewing yeast strain is used in fuel industry.WO2005/111214 discloses the Wine brewing yeast strain producing the ability of ethanol under inhibitor (as furfural and 5-methoxyl group furfural) exists with increase.
Different with several yeast species from bacterium, wild type Saccharomyces cerevisiae can not use pentose (as wood sugar and pectinose) as carbon source.Yeast saccharomyces cerevisiae utilizes and enriches the ability that carbon source (as from the effluent of other techniques and waste material, as from the agricultural waste of such as corn and bagasse and the waste material from such as papermaking) grows and have huge environmental value and also have economic worth.Agricultural wastes comprise quite most hemicellulose, and this hemicellulose comprises many different sugar monomers.For example, in addition to glucose, these sugar monomers can also comprise wood sugar, seminose, semi-lactosi, rhamnosyl and pectinose.Wood sugar is the maximum sugar monomer of content and therefore represents a kind of important carbon source for using yeast to manufacture ethanol, provides huge economy and environment advantage.
There is provided and use wood sugar to be previously introduced in yeast saccharomyces cerevisiae as the gene encoding enzyme of the ability of carbon source.EP1282686 discloses the gene for enzyme Xylose reductase, xylitol dehydrogenase and xylulokinase that has and be merged in and by the recombinant Saccharomyces cerevisiae bacterial strain of specific mutant.It is the ability of ethanol that described bacterial strain has lignocellulosic material fermentation.In EP1282686, the bacterial strain of institute's preservation is CBS102679(TMB3400, Taurus01) and be considered to effective in the prior art generally.It is extraordinary that the ethanol produced by bacterial strain CBS102679 has been considered to compared with other prior art recombination yeasts, but also produces undesirable by product Xylitol.Therefore, in view of the environmental problem that society increases gradually, in this area, still need to provide the novel Wine brewing yeast strain with even better ethanol production, better xylose rate and lower Xylitol output.In addition, desirable to provide the bacterial strain with even better inhibitor tolerance.
summary of the invention
Therefore, the object of the invention is to solve the problem and provide the novel Wine brewing yeast strain of inhibitor tolerance with better xylose rate, better ethanol production, lower by product Xylitol output and improvement, described Wine brewing yeast strain has and substantially only uses wood sugar as carbon source to produce the ability of ethanol.
According to a first aspect of the present invention, a kind of method for generation of Wine brewing yeast strain is used to solve this problem, this Wine brewing yeast strain has the gene of encodes xylose reductase, xylitol dehydrogenase and the xylulokinase be introduced into, and have the ethanol production of improvement, the xylose rate of improvement and the Xylitol output of reduction, this method comprises the following steps:
A) in a continuous mode, substantially the substratum of wood sugar as carbon source is only comprised with a kind of, under 25 ° of C-38 ° of C, the preferably temperature of 30 ° of C-35 ° of C and the air-flow of 0.01vvm-0.06vvm, cultivate the brewing yeast cell with the gene of encodes xylose reductase, xylitol dehydrogenase and the xylulokinase be introduced into, and increase thinning ratio to maintain constant cell level, described cell levels is determined within the scope of 1.5-3.0 by optical density (OD) or equivalent analysis means
And to add at least one inhibitor to these cells and to increase the interpolation of described inhibitor gradually b).
Important step is that the cell levels maintained in bio-reactor is constant.When the cell of improvement of evolving out, these cells improved more preferably can utilize substrate with the particular growth speed set by thinning ratio, and the result of improvement is by more for formation cell.This means, for keeping selective pressure, to need growth velocity to increase, growth velocity increase obtains by increasing thinning ratio.
According to one embodiment of present invention, step this substratum a) comprises the wood sugar of wood sugar, the preferably about 20g/l of 15g/l-25g/l.
In yet another embodiment, the method comprises step in addition
C) in the training period, such as, after step a) or b), temperature be increased to 35 ° of C-45 ° of C and/or apply uv-radiation.
The number of the cell for stress with more height endurability has been enriched in the increase of temperature.
Air-flow used depends on the characteristic of used yeast bacterial strain.Under respiratory tract metabolic condition, require more air, such as, in xylose metabolism situation.Start once ethanol produces, then require less air and may reduce to make the selective pressure of alcohol metabolism increase on the contrary.
Wood sugar consumes ability and may be regarded as being made up of two kinds of integral parts, input rate and can effectively inputting under which kind of concentration, namely to the avidity of wood sugar.When wood sugar is sole carbon source, input rate and growth velocity are closely related, and how soon what namely wood sugar curve reduced has.If wood sugar curve arrives close to zero, namely all wood sugars are used all, and so wood sugar avidity is good.
For effective xylose ability, the carbon of inputted wood sugar should to be imported in addition in ethanol instead of in Xylitol and the least possible guiding cell formed.Bacterial strain of the present invention has input rate and the wood sugar avidity of improvement.
In another embodiment, by the Xylitol output of the ethanol production improved, the xylose rate of improvement and reduction with CBS102679(TMB3400, Taurus01) bacterial strain of preservation compares.In another embodiment, as aforesaid method step a) in these cells used cell that is the bacterial strain of the CBS102679 of such as institute's preservation.Step a) in be used as parent material described brewing yeast cell there is the gene for enzyme Xylose reductase, xylitol dehydrogenase and xylulokinase be introduced into, to enable this primary yeast xylose-fermenting.These genes can obtain from any source that can be separated these genes.For example, the gene of encodes xylose reductase, xylitol dehydrogenase can obtain from pichia stipitis (Pichiastipitis), and the gene of encoding xylulokinase can obtain from yeast saccharomyces cerevisiae.
In one embodiment, the method comprises step in addition
D) from step b) or from step c) select the cell with xylose ability and inhibitor tolerance.
According to another embodiment, carry out this selection as on the agar plate of sole carbon source and/or described at least one inhibitor or any other inhibitor substantially having wood sugar.Also likely select on the agar plate with any carbon source and described at least one inhibitor or any other inhibitor.Therefore, this bacterial strain can tackle the environment with dissimilar inhibitor, these inhibitor be not only in the inventive method to make bacterial strain have the specific one of inhibitor tolerance.
For obtaining the inhibitor tolerance wood-sugar fermentation yeast strain of improving, importantly keep selective pressure and the inhibitor tolerance of xylose.Because glucose is a kind of preferred yeast saccharomyces cerevisiae carbon source, so the existence of this sugar inhibits the selective pressure of wood sugar.This has reduced in some experiments of xylose ability and has been noted while obtaining inhibitor tolerance.Therefore, the hydrolysate of most of type can not be used for according in method of the present invention (typically comprising a large amount of glucose).Therefore, in order to produce a kind of bacterial strain of the present invention, a kind of laboratory culture base (minimum medium or synthetic medium) should be provided, this substratum has wood sugar as the sole carbon source substantially under inhibitor (as synthetic mixture) existence or the hydrolysate as principal degradation, high and the sugared content of inhibitor content of this hydrolysate is low, and importantly the level of glucose and seminose should be very low.
In one embodiment of the invention, this inhibitor is selected from furans, as HMF and furfural; Organic acid, as acetic acid and formic acid; And phenol system compound.The other example of inhibitor provides in the sample portion of this specification sheets.
The present invention relates to a kind of Wine brewing yeast strain in addition, and this Wine brewing yeast strain has the Xylitol output of the ethanol production of improvement, the xylose rate of improvement and reduction, the Taurus03 of wherein said bacterial strain to be preserving number be CBS128138.
In another embodiment, the invention still further relates to a kind of Wine brewing yeast strain, this Wine brewing yeast strain has the ethanol production of improvement, the xylose rate of improvement and the Xylitol output of reduction and the inhibitor tolerance of improvement, the Taurus04 of wherein said bacterial strain to be preserving number be CBS128139.
In one embodiment, the invention still further relates to a kind of Wine brewing yeast strain, this Wine brewing yeast strain has the ethanol production of improvement, the xylose rate of improvement and the Xylitol output of reduction and the inhibitor tolerance of improvement, the Taurus07 of wherein said bacterial strain to be preserving number be CBS128140.
All bacterial strains of the present invention have been deposited in fungal organism center of diversity (the CBS) (CentraalbureauvoorSchimmelcultures (CBS) of No. 8, Uppsala, Dutch 3584CT Utrecht city all, Uppsalalaan8,3584CTUtrecht, theNetherlands).
Preserving number is that the yeast saccharomyces cerevisiae Taurus03 of CBS128138 is in preservation on October 26 in 2010.
Preserving number is the yeast saccharomyces cerevisiae Taurus04 of CBS128139, preserving number is that the yeast saccharomyces cerevisiae Taurus07 of CBS128140 is in preservation on October 26 in 2010.At this yeast saccharomyces cerevisiae Taurus10CBS128141 also mentioned in preservation on November 2 in 2010.
An alternative embodiment of the invention is the purposes of a kind of bacterial strain for being ethanol obtained by the inventive method by lignocellulosic material fermentation.Lignocellulosic material used can be operational any classification.Example is agricultural residue, comprises maize straw and bagasse; Wood residues, comprises sawmill and paper mill Litter; And city paper using waste.
An alternative embodiment of the invention is a kind of bacterial strain of being obtained by the inventive method purposes at the same time in saccharification and fermentation (simultaneoussaccharificationandfermentation, SSF) technique.
brief Description Of Drawings
Now describe the present invention in detail by example reference institute accompanying drawings.
Fig. 1: the improvement of the growth characteristics reflected by thinning ratio between the adaptive phase in the cultured continuously of use minimum medium described in example 1.In first part, (blue line originates in 0.025 thinning ratio, h
-1) the middle wood sugar that uses is as sole carbon source, and (red line, originates in 0.05 thinning ratio, h at second section
-1) in temperature is increased to 35 ° of C and (green line originates in 0 thinning ratio, h by bagasse with the level increased
-1) be blended in substratum.
Fig. 2: the improvement of maximum particular growth speed between the adaptive phase in the shake-flask culture repeatedly using minimum medium described in example 2.First part's (rhombus) has the wood sugar of 20g/l and second section (square) has 5g/l.In the second section of taking turns from the 10th, show serial D.
Fig. 3: at use minimum medium and wood sugar as the wood sugar consumption (square) in the shake-flask culture of sole carbon source and ethanol (cross) and cell (rhombus) formation.Cell concn is with optical density (OD) (OD) the form assessment under 650nm.Fig. 3 a is Taurus02, Fig. 3 b be Tarurus03, Fig. 3 c be Taurus07, Fig. 3 d be Taurus04, Fig. 3 e be Taurus08, Fig. 3 f be Taurus09, Fig. 3 g is Taurus10.
Fig. 4: the glucose (square) in using the anaerobic biological reactor of minimum medium to cultivate and the formation of wood sugar (trilateral) consumption and biomass (rhombus), ethanol (cross) and Xylitol (star).Representative cultivation is shown in duplicate.Fig. 4 a is Taurus01, Fig. 4 b be Tarurus03, Fig. 4 c be Taurus09, Fig. 4 d is Taurus10.
Fig. 5: by first-generation wood sugar bacterial strain (Taurus03(rhombus) and Taurus09(trilateral in the anaerobism shake-flask culture using corn cob liquid)) the wood sugar consumption carried out, respectively with the s-generation wood sugar bacterial strain (Taurus04(is square) of being evolved by these bacterial strains and Taurus10(cross)) compare.
Fig. 6: in the SSF experiment of the cellulose degrading enzyme of the birch slurry and 5FPU/gWIS that utilize 7.5%WIS, substrate glucose (rhombus), wood sugar+seminose+semi-lactosi are (square, Xyl-Man-Gal), product ethanol (circle), Xylitol (cross), and furans inhibitor HMF(star) and the concentration curve of furfural (trilateral).Fig. 6 a is Taurus01 and 6b is Taurus03.
Fig. 7: use Taurus04 initial after 2h and use the corn cob liquid of glucose charging batch feed fermentation in, the curve of the total amount of glucose (rhombus), semi-lactosi (trilateral), wood sugar (square), ethanol (black circles) and Xylitol (orange circle).
Fig. 8: there is the serial dilution series that the YPD Agar plate of the mixture of 12 kinds of inhibitor is tested.Each circle corresponds to once dilution and extension rate is larger more to the right.
Fig. 9: the anaerobically fermenting using the bagasse hydrolysate of yeast saccharomyces cerevisiae Taurus03, Taurus04, Taurus07 and Taurus10 in shake-flask culture.
Figure 10: utilize wheat stalk slurry to use SSF(synchronous glycosylation and the fermentation of Taurus07) experiment.
Figure 11: in a kind of Industrial demonstration scale fermentation equipment, in the hydrolysate of corn cob leftover slurry, (Figure 11 a) and at corn cob leftover slurry uses the fermentation of Wine brewing yeast strain Taurus04 in conjunction with (Figure 11 b) in SSF technique.
Figure 12: utilize the acetate of wood sugar growing period to different concns and the tolerance of HMF at bacterial strain Taurus01, Taurus03, Taurus04, Taurus07, Taurus09 and Taurus10.
Figure 13: (Figure 13 a), uses the consumption/generation curve (Figure 13 b) in the fermentation of the C6 solid through hydrolysis of Taurus04 and uses the consumption/generation curve (Figure 13 c) passed through in the fermentation of the C6 solid of hydrolysis of Taurus04 the sugar consumption in using the C5 of Taurus04 to ferment.
Figure 14: use the C5 material of Taurus04 bacterial strain and the synchronous glycosylation of C6 solid and fermentation
detailed description of the preferred embodiment
When carrying out experiment of the present invention, except as otherwise noted, otherwise use conventional microbiological technique.This type of technique is known to persons of ordinary skill in the art and is fully explained in the literature.
In addition, all technical terms used in the application all have usual the understood implication of those of ordinary skill.
In such as plant biomass, wood sugar is abundant and use yeast (as yeast saccharomyces cerevisiae) to utilize wood sugar to cause further investigation in this technical field as the possibility that carbon source produces ethanol.But xylose rate is sometimes bad, thus cause bad ethanol production.In addition, the output of by product Xylitol is quite large.
Ladies and gentlemen contriver of the present invention unexpectedly develops the Wine brewing yeast strain with more effective xylose and the therefore improvement of better ethanol production in view of the above problems.In addition, lower by product Xylitol output has been observed.In addition, these bacterial strains improved have better tolerance for the various inhibitors found in many wood fibre hydrolysis products.
It should be noted that and wood sugar can be used also can to utilize other sugar growths as the bacterial strain of sole carbon source substantially.The implication of phrase " substantially sole carbon source " means other sugar that can also there is trace.Glucose can be there is and it is usually first by Saccharomyces cerevisiae transformant, because it is preferred sugar concerning yeast.After this, use wood sugar as carbon source.
Used known preserving number to be CBS102679 be called the Wine brewing yeast strain of XYLUSM125, TMB3400 or Taurus01 achieve yeast saccharomyces cerevisiae these desired by feature.In this bacterial strain, introducing coding and the gene using wood sugar as the enzyme of the ability of sole carbon source is substantially provided.Alternately, other Wine brewing yeast strains having and use wood sugar as the ability of sole carbon source substantially can be used according to the present invention.These bacterial strains of the present invention can with the form n-back test of industrial strain and laboratory strains, even if industrial strain is preferred.Industrial strain is more bad compared with laboratory strains to be determined, because it has each several copy chromosomal.Therefore, the operation industrial strain as carried out according to the present invention is a larger challenge compared with operation laboratory bacterial strain.
According to of the present inventionly having the ethanol production of improvement, the bacterial strain of the xylose rate of improvement and the Xylitol output of reduction and the inhibitor tolerance of improvement uses non-recombinant method to obtain.This means not utilize recombinant DNA technology to obtain there is improvement ethanol production, the xylose rate of improvement and the Xylitol output of reduction and improvement the bacterial strain of inhibitor tolerance.But, use recombinant DNA technology to obtain the bacterial strain TMB3400(Taurus01 that such as can be used as according to parent material of the present invention).Recombinant DNA technology according to the present invention refers to the multiple technologies in vitro operating genetic information, but not recombination method does not utilize and thisly in vitro to operate.
According to the present invention, achieve desired feature, (usually occur at growing period and may increase because of uv-radiation) is selected and enrichment, i.e. orthogenesis/adaptation or evolution engineering because suddenly change during specified conditions in the cultivation of yeast saccharomyces cerevisiae.The program of orthogenesis can be divided into three steps: first make sudden change occur, and after this selects desired proterties by applying selective pressure in cultivation, and finally according to the bacterial strain that figure spectral property screening/sign obtains.
Sudden change is there is, because some mistakes may be there are during DNA is replicated and transfers to the reproduction process in the next generation in genetic code during normal growth.The probability that sudden change occurs can increase because of some chemical or uv-radiation.These sudden changes can change the characteristic of cell protein, make the possibility of organisms exist increase or reduce.
Term " selection " refers to " adaptation " process, in this process, has cell enrichment in colony of the desired feature of improvement.This realizes by making desired characteristic apply selective pressure to the survival of microorganism and beneficial properties by design growth conditions in cultivation, and this selective pressure can be selected from conditions different in a large number.As for the present invention, selective pressure can by making wood sugar as sole carbon source substantially, making inhibitor exist or increase temperature to realize.In addition, different selective pressures is realized by training mode.Temperature increase can make the cell enrichment with higher integrated stress tolerance.It is highly important that the suitable selective pressure selecting and apply to allow to be formed gradually desired characteristic.In identical adaptation or in follow-up cultivation, some selective pressure can be applied together.Typically, according to the present invention, in liquid medium within or can cultivate on a lbmc agar plate.In liquid culture, increase in the ratio adapting to the cell improved between progressive stage and be regarded as the improvement of the performance of population mixture, as the present invention, this improvement is reflected by the xylose rate of the ethanol production improved, improvement and the Xylitol output of reduction.
Importantly, should proceed to adapt to during many generations, to allow to occur applicable sudden change and enrichment in cell colony.When selecting on a lbmc agar plate, be typically enough to make cell to have growth and the time durations forming the ability of bacterium colony cultivates cell.The larger bacterium colony instruction occurred on a lbmc agar plate has the cell of the ability grown under the applied conditions of improvement, and comparatively speaking, less bacterium colony shows poor ability.
In this article, phrase " a new generation " means that cell as one of ordinary skill in the understanding has been divided into two new cells and these two new cells represent a new generation.In this article, in cell continued growth many generations and until no longer can observe any character mutation compared with earlier generations in a new generation, namely character mutation keeps constant.The example of character mutation is the maximum particular growth speed of such as cell.This can measure by measuring optical density (OD).Other character mutation that can observe are the output of such as alcohol production rate, xylose rate and by product Xylitol.When these integral parts remain on constant level, no longer observe character mutation.
In addition, term " screening " refer to a kind of with can manner of comparison by the process of the performance characterization of cell.Screening method should show the performance of the desired characteristic of reflection cell and can carry out on a lbmc agar plate or in liquid medium within.Therefore, can identify the desired feature with improvement cell and for the further sign of other characteristics.
Therefore, according to the present invention, provide a kind of method for generation of Wine brewing yeast strain, this Wine brewing yeast strain has the gene of encodes xylose reductase, xylitol dehydrogenase and the xylulokinase be introduced into and has the ethanol production of improvement, the xylose rate of improvement and the Xylitol output of reduction and the inhibitor tolerance of improvement.
In addition, provide by the obtainable multiple Wine brewing yeast strain of the method according to this invention.
It is hereafter the example for obtaining the Wine brewing yeast strain according to improvement of the present invention.All working is all with bacterial strain TMB3400(Taurus01) initial, this bacterial strain had previously shown the extraordinary performance about xylose ability and quite low Xylitol output (people such as Song Deleige (Sonderegger), 2006, Hahn-Ha Gedaer
deng people, 2007).But, as stated previously, also can use other Wine brewing yeast strains, as long as it has the ability of xylose-fermenting.By the present invention, unexpectedly likely produce and produce even more effective Wine brewing yeast strain about xylose and ethanol.Also observe lower by product Xylitol output.Therefore, as according to the present invention the bacterial strain that obtains to may be used for fermenting different lignocellulosic materials or the waste material from corn or bagasse, these materials also comprise quite a large amount of wood sugars in addition to glucose.Await will there is huge economic worth and environmental value for the more effective bacterial strain in this type of zymotechnique.
As will be visible in experimental section, bacterial strain of the present invention demonstrated compared with prior art bacterial strain produce about ethanol, wood sugar consumption, Xylitol produce and the superiority of inhibitor tolerance.
example 1
use the xylose ability of cultured continuously and the improvement of inhibitor and temperature tolerance
Use the initial directional adaptation series of bacterial strain Taurus01 and at a bio-reactor (about 750ml working volume in 1l, Bei Laqi biotech company of Sweden (BelachBioteknikAB, Sweden) carry out in cultured continuously), this bio-reactor has the minimum medium (people such as Wilden, 1992) as defoamer (in bioreactor culture) of the PEGP2000 compared with used according to Wilden (Verduyn) with 2 times of high trace metal solutions and 0.1ml/l.With 2MNaOH pH value controlled 5.0 and agitator speed is set in 350rpm.For initial cultivation, 50ml had the inoculum of 20g/l wood sugar and 20g/l glucose (in 250ml shaking culture bottle (baffledshakeflask), cultivate 24h-36h, 30 ° of C, vibrate under 180rpm) add bio-reactor to and (there is minimum medium and 20g/l wood sugar and 20g/l glucose, cumulative volume 800ml, flows into without gas) in.Carry out cultivating lasting 15h-20h, then initial continuous mode, wherein make only there is 20g/l wood sugar as the substratum inflow of carbon source and with a upflow tube mensuration discharge.Accurate culture volume is measured and for 740ml-780ml at the end of cultivation.Optical density (OD) (OD) under measuring 650nm each working days is measured as of cell density.Regular acquisition is for measuring the sample (also from culture flash) of extracellular metabolite and needing the aliquots containig of cell of freezen protective (adding 1ml cell suspending liquid in aseptic 60% glycerine of 0.5ml, under being stored in-80 ° of C).Use HPLC(Dai An company (Dionex), Ultimatum3000, under 35 ° of C, specific refractory power detects (RIdetection), at 210 nm ultraviolet detection, the tubing string from Bole company (BioRad) under 45 ° of C; AminexHPX-87H and 30mm × 4.6mm positively charged ion-H Mike Luo-Jia De (micro-guard), eluent 5mMH
2sO
4, under the flow velocity of 0.6ml/min) and measure metabolite.Use microscope eyes to make regular check on culture, find to there is not pollution.
The first part adapted to carries out under the air-flow of 30 ° of C and 35ml/min.Object is value OD being remained on 2-2.5.OD level increases the improvement of instruction growth characteristics and therefore can increase flow velocity to apply higher selective pressure.Accordingly, if see OD level reduce, so selective pressure too high and therefore need reduce flow velocity.Therefore, flow velocity increase (or recalculating according to thinning ratio) be cell performance increase one measure.After 15 generations, temperature in reactor is increased to 35 ° of C-45 ° of C and continues 24h, and from culture, reclaim cell, preserve freezing aliquots containig and a sample is above rule at a wood sugar agar plate (20g/l wood sugar, 20g/l peptone, 10g/l yeast extract and 20g/l agar).Be Taurus02 by the Strain Designation obtained after this Temperature Treatment.Use the initial new cultured continuously of cell from agar plate.Carry out cultivation and continued for 77 generations, visible thinning ratio increases (Fig. 1) and is Taurus03 by last Strain Designation in the meantime.Therefore, this bacterial strain belongs to first-generation wood sugar bacterial strain.
The second section adapting to series carries out under the air-flow of 13ml/min and applies other selective pressure.First, the temperature in bio-reactor is increased to 35 ° of C(in batches all like this with successive stage) and secondly with the level increased, poisonous bagasse is blended in substratum.Pre-treatment is used (to use 2%SO
2about 190 ° of C) prepare this bagasse hydrolysate (at SEKABE-technology company of Sweden (SEKABE-technology, Sweden)), the long period (residence time 13min-15min) is carried out in this pre-treatment, produces the hydrolysate of slightly degrading with low sugar level (4g/l glucose, 5g/l wood sugar, 0.4g/l semi-lactosi) and high inhibitor content.When preparing substratum at every turn, with 5MNaOH, the pH value of the hydrolysate having aliquots containig to be used is adjusted to 5.0.First only continuing for 21 generations to allow cell adapted higher temperature containing the minimum medium of wood sugar carrying out cultivate.Then carry out cultivation and continue 120 generations altogether.Between cultivation progressive stage, visible thinning ratio increases, but growth is slower when increasing the bagasse content in substratum, and therefore needs to reduce thinning ratio (Fig. 1).But, along with these generations continue carry out, under a certain bagasse content, visible thinning ratio improves (Fig. 1).
At the end of orthogenesis scheme, obtain s-generation wood sugar bacterial strain and characterize further.Latter two bacterial strain (called after Taurus05 and Taurus06) is used to select the specific inhibitor tolerance of the heterogeneous cell population obtained between the adaptive phase to clone for Taurus04 third from the bottom Strain Designation.With sterilized water with the sample of ten times of dilution series diluting cells.Each diluent 10 μ l is dropped in one have on the agar plate (20g/l glucose, 20g/l peptone, 10g/l yeast extract, 20g/l agar) of the synthetic mixture of inhibitor selected by 12 kinds.These inhibitor exist with following concentration: 2.5g/l hydroxymethylfurfural, 0.82g/l furfural, 4.7g/l acetic acid, 0.9g/l formic acid, 1.8g/l levulinic acid, 0.10g/l Vanillin (vanillin), 0.03g/l coniferyl aldehyde (coniferylaldehyde), 0.75mg/l styracin, 15mg/l quinhydrones, 82mg/l syringic aldehyde (syringaldehyde), 15mg/l4-hydroxy-benzoic acid and 15mg/l4-hydroxy 3-methoxybenzene benzylacetone (guaiacyl acetone (guaiacylacetone)).Cultivate these plates until larger bacterium colony.By the line and cultivate two days under 30 ° of C and at room temperature cultivate four days again on bagasse board (50% poisonous bagasse, 20g/l glucose, 20g/l peptone, 10g/l yeast extract, 20g/l agar) of the cell (bacterium colony be Taurus05,3-4 bacterium colony be Taurus06) from these bacterium colonies.Several larger bacterium colonies are rule on new bagasse board again and cultivates 2+4 days again.By six of each bacterial strain from these plates maximum bacterium colonies respectively at xylose minimal medium lining out.Cultivate under 30 ° of C after seven days, use the 9-10 of each bacterial strain maximum bacterium colony initial small incubation (10ml is in 50ml centrifuge tube) in the substratum (20g/l wood sugar, 20g/l peptone, 10g/l yeast extract) being rich in wood sugar, these colony growths are spent the night and subsequently for the preparation of freezing cell storing solution, produces the bacterial strain of called after Taurus07 and Taurus08.
example 2
use repeatedly the improvement of the xylose ability of shake-flask culture
Using from TMB3400(Taurus01) the Taurus02 bacterial strain that develops into is as the initial strain of directional adaptation scheme using Repeated batch process.In shake-flask culture, under the stirring of 30 ° of C and 180rpm, there is the basic xylose media of the 50ml (people such as Wilden, 1992) cultivate in 100ml flask, this basic xylose media has pH5.5 and the metallic solution of 2 times of high traces compared with used according to Wilden.The first part of adaptation scheme uses 20g/l wood sugar to carry out in the medium.Cultivate each time until when grow start to stop time 650nm under optical density (OD) (OD) reach the value of 1.8-3, that then the aliquots containig of cell is transferred to next round has in the flask of fresh culture, obtains the initial OD of 0.07.In taking turns the 7th, performance is improved to a certain degree, and the initial OD making the 8th to take turns is reduced to 0.06, and 9-10 wheel is 0.05 and 11-13 wheel is about 0.02.Altogether carry out 13 times to cultivate, during adaptation scheme, allowed for 73 generations and 7 cell freezings of taking turns preserve (being added to by 1ml cell suspending liquid in aseptic 60% glycerine of 0.5ml, under being stored in-80 ° of C) in these being taken turns.Measure OD after these are cultivated and calculated maximum particular growth speed (Fig. 2) by these OD and measure cultivate each time time and extracellular metabolite when transferring to next round.Use HPLC(Dai An company, Ultimatum3000, under 35 ° of C, specific refractory power detects, ultraviolet detection at 210 nm, the tubing string from Bole company under 45 ° of C; AminexHPX-87H and 30mm × 4.6mm positively charged ion-H Mike Luo-Jia De, eluent 5mMH
2sO
4, under the flow velocity of 0.6ml/min) and measure metabolite, and during these results display transfer, wood sugar is on close level height, about 12g/l-16g/l, and therefore this adaptation is carried out to next section.Last Strain Designation is Taurus09 and therefore together with Taurus03 for another kind of first-generation wood sugar bacterial strain.This bacterial strain has the maximum particular growth speed (Fig. 2) of improvement about three times under condition of compatibility.
According to the performance of bacterial strain Taurus09 difference in the substratum with 5g/l wood sugar, and be all chosen as compared with 10g/l in independent cultivation, in the second section of adaptation, xylose concentration be chosen as 5g/l.In this adaptation, under OD2.8-3, carry out cell transfer and initial OD is 0.1(except initial OD is the initial four-wheel of 0.01) and follow the trail of OD and metabolite as previously mentioned.Proceed to adapt to during corresponded to for 47 generations 9 take turns.Because the particular growth speed seen at this moment improves limited (Fig. 2), so culture to be divided into four parts.Two part UV-irradiation are to increase sudden change number, and two parts are just transferred in next round without any process.For ultraviolet process, use the agar plate with basic xylose media, and cell is spread in 4 on the agar plate of super-dry.Using makes two plates be exposed to UV-light from TFM-26V, a P/N95-0422-02 of ultraviolet Products Co., Ltd (Ultra-VioletProductsLtd.), 25 watts of effective UV transilluminators, its medium wavelength is 302nm, be set in high strength, continue 60 seconds and two plates continue 90 seconds.Collecting cell and for the new cultivation in initial adaptation series from these plates immediately.The cell one of exposure 60 and 90 seconds is used from new shake-flask culture.Ultraviolet process and undressed cell are all used for initial cultivation under normal condition and the limited condition of oxygen, see table.
The limited condition of these oxygen realizes by using a special shaking flask.The limited flask of this oxygen is made up of a 250ml shaking flask with multiple plate washer and two arms.An air filter (strainer 21978 from Yi Fusen company (INFORSHT)) is connected with one of these arms, and a rubber hose in external end with a syringe is connected for sampling with another arm.All openings all seal with Parafilm.These are adapted to series and carries out 14-15 wheel, except the serial B of necrocytosis in taking turns second.The number in the generation obtained during this final section is series A 83, serial C68 and serial D89.Period regularly Cell Cryopreservation (preparing as described above) is proceeded in adaptation.The last bacterial strain of series D shows optimum performance and is selected as s-generation wood sugar bacterial strain and called after Taurus10.This bacterial strain has the maximum particular growth speed (Fig. 2) of improvement about 5 times in adaptation under low wood sugar level.
example 3
bacterial strain is about the sign of wood-sugar fermentation ability at different conditions
a) use minimum medium and wood sugar as the shake-flask culture of sole carbon source
The sign of the first kind applied be use minimum medium and only wood sugar as the shake-flask culture of carbon source.Carry out cultivating in 50ml substratum in 100ml flask and pass through add initial from the cell of freezing storing solution, obtain 0.01 OD.At 30 ° of C and 180rpm(, this produces half aerobic condition in these flasks) under cultivate culture, and follow the trail of cell and metabolite concentration.
Carry out the bacterial strain of this type sign:
·Taurus02
·Taurus03
·Taurus07
·Taurus08
·Taurus09
·Taurus10
First-generation wood sugar bacterial strain (Taurus03 and Taurus09) from the orthogenesis scheme of two types has achieved maximum particular growth speed faster, and s-generation wood sugar bacterial strain only slightly improves (table 1, Fig. 3).These bacterial strains have the metabolic type to ethanol generation skew and therefore form less cell.Therefore, can infer, consider that the bacterial strain of all improvement all can use the wood sugar of greater part in addition, wood-sugar fermentation is more effective.Optimum strain Taurus10 in this respect can use the wood sugar more than 90% and therefore this bacterial strain produces the highest levels of ethanol 4.2g/l.Wood-sugar fermentation difference is not found between inhibitor resistant strain Taurus07 and Taurus08.
Table 1. use minimum medium and wood sugar as sole carbon source shake-flask culture during growth velocity, output, the residual level of wood sugar and maximum concentration of ethanol.Taurus02, Taurus07, Taurus10 carry out with the form of cultivating in duplicate, and Taurus03 is triplicate, and these results of cultivating provide with the form of a scope.
All bacterial strains all form Xylitol with low-level (often consuming one gram of wood sugar <0.06g)
b) mineral medium and glucose and xylose is used to cultivate as the anaerobic biological reactor of carbon source
In a kind of industrial setting, anaerobism performance is extremely important and therefore characterize in the cultivation of this type.Because cell only can not utilize wood sugar to grow (Fig. 4) as carbon source in anaerobism mode, so use the mixture of glucose and wood sugar.
Pre-culture is prepared by being added in the 100ml minimum medium comprising 20g/l glucose and 20g/l wood sugar from the frozen cell of stock culture by 100 μ l.Then under 30 ° of C, cultivate culture 48h in aerobic mode, then bio-reactor is inoculated.The culture medium supplemented of Anaerobic culturel will be used for 0.1ml/l defoamer (polypropylene glycol P2000), 10mg/l ergosterol and 0.42g/l tween 80 (Tween80) in bio-reactor.Use working volume be two liters bio-reactor (Bei Laqi biotech company of Sweden) and by nitrogen being realized anaerobic condition by fermentation container with 0.4l/min.Temperature is controlled at 30 ° of C, make pH value be 5.0 with 2MNaOH, and agitator speed is 500rpm.Then the cell from pre-culture is added, to reach the initial OD of 0.01 in bio-reactor
650.During fermentation process periodic measurement OD and dry cell wt and the outer metabolite sample of collecting cell.Each bacterial strain is cultivated in duplicate, and these cultivations obtain very similar result.
Carry out the bacterial strain of this type sign:
·Taurus01
·Taurus03
·Taurus09
·Taurus10
Or during anaerobic condition, for all improvement bacterial strain wood-sugar fermentation all more effectively (table 2, Fig. 4).Both all visible wood sugar wear rates increase and the bacterial strain that all wood sugars all can be modified consumed.Particularly, bacterial strain Taurus10 shows consumption fast.Ethanol with the improvement by our bacterial strain realization forms being formed of the by product Xylitol being also shown in reduction.For Taurus03, the reduction of Xylitol output reaches 40%.
Table 2. has xylose and glucose as the output on the minimum medium of carbon source and wood sugar consumption in Anaerobic culturel.Provide duplicate maximum deviation of cultivating.
Bacterial strain of the present invention (Taurus03, Taurus09 and Taurus10) has higher alcohol concn and lower Xylitol output compared with well-known Taurus01.
c) anaerobically fermenting of corn cob liquid in shake-flask culture
About the next step in sign, assessed the performance in actual industrial substrate by the fermentation of research in corn cob hydrolysate.By with SO
2form add dilute acid pretreatment and make corn cob by SEKABE-technology company of Sweden.Filter pretreated slurry to obtain liquid portion.
In preculture, described in b, make cell grow on the minimum medium with glucose and xylose, after 24h, some corn cob liquid to be added in shaking flask and to make it cultivate 18h again.Pass through centrifugal collecting cell.For fermenting, using and being supplemented with 0.5g/l (NH
4)
2hPO
4corn cob liquid as substratum, pH value is set as 6.0.Add the cell collecting (by centrifugal) from pre-culture, obtain the approximate initial cell density of often liter of 3g dry weight.Use 50ml substratum in 100ml shaking flask, under 30 ° of C and 180rpm, carry out the cultivation of culture, and by using the vent plug of filling with glycerine to obtain anaerobic condition.Metabolite sample is obtained after 0h, 2h, 4h, 6h, 8h, 24h, 48h, 72h and 96h.
Carry out the bacterial strain of this type sign:
·Taurus03
·Taurus04
·Taurus09
·Taurus10
Table 3 and the data in Fig. 5 clearly show that s-generation wood sugar bacterial strain Taurus04 with Taurus10 compares the higher final alcohol concn of generation and xylose rate with their respective parent strain Taurus03 with Taurus09 after 96h.Although xylose rate is large, the output of by product Xylitol remains on low-level (Fig. 5).
The Product yields of table 3. in the anaerobism shake-flask culture utilizing corn cob liquid, wood sugar consumption and final levels of ethanol.Carry out single cultivation.
d) SSF(synchronous glycosylation and the fermentation of corn cob and birch slurry is utilized) experiment
The another kind of typical process that ethanol produces is SSF technique.In this technique, lytic enzyme adds together with yeast, to realize the fermentation of cellulosic decomposition in pretreated slurry and monomer sugar simultaneously.In the process, substrate and processing step has been used to simulate a kind of industrial situation as much as possible.Cell proliferation is carried out in the batch feed culture of ventilation.Preculture for breeding is carried out in the 50ml minimum medium (pH6.0) with 20g/l glucose and 20g/l wood sugar in 150ml shaking flask.With sub-fraction (cell that 100 μ l are freezing) this preculture initial and in a gyrate shaker, under 30 ° of C, carry out best 24h(18h-36h).In this batch, substratum (500ml) comprises 50g/l molasses and 23.5g/l (NH
4)
2sO
4, 3.0g/lKH
2pO
4, 2.25g/lMgSO
47H
2o and 99 μ g/l vitamin Hs, 360ppm tie up his hops (Vitahop) (the hops extract for anti-bacteria) and 1.2ml/l defoamer (based on silicon, Nuo Puke ENA-309(NopcomasterENA-309), Nuo Puke Zhi Ye technology company (NopcoPaperTechnologyAB)).Adding whole pre-culture (inspires confidence in gloomy bio-reactor (Labforsbioreactor) at Lai Bu with initial cultivation, Yi Fusen company (InforsAS), this cultivation carries out under the following conditions: with 1vvm ventilation, with 3MNaOH, pH value is adjusted to 5.0, and stirring velocity is 700.Carry out this batch until sugar runs out (about 8h-10h) and then initial charge.Be about in the SSF carried out, use the charging (pH value is adjusted to 5, is diluted to corresponding to 7.5%WIS) comprising the hydrolysate of material, and comprising molasses, thus producing total hexose sugar concentration of 75g/l.When the batch feed stage is initial, agitator speed is increased to 1000rpm and ventilation be increased in final volume body 1.0vvm.Charging is with 0.1h
-1constant rate of speed increase and carry out 20h, thus produce the final volume of 1.5l.By centrifugal (under 1800 × g 8min) collecting cell and settling flux in aseptic 0.9%NaCl.Utilize 7.5%WIS and be supplemented with 0.5g/l (NH
4)
2hPO
4, the 125ppm slurry (pH5.0 regulates with NaOH) of tieing up the pretreated ligno-cellulosic materials of his hops and 0.4ml/l defoamer (identical with breeding) carries out SSF part (total substratum 1.5kg).Add the cell of settling flux in 0.9%NaCl, obtain the initial cell concentration of often liter of 3g dry weight, and add enzyme (NS-22074 or CellicC-tec2, Novi's letter (Novozymes)), obtain 5FPU/gWIS with this technique initial.Controlled, at 35 ° of C, with 3MNaOH, pH value to be adjusted to 5.0 by fermentation container (not containing plate washer, Lai Bu inspires confidence in gloomy), agitator speed be about 300rpm-400rpm, and the outlet that all entrances are all closed and pass condenser is opened.At least obtain the sample being used for metabolite analysis in the following time: 0h, 2h, 4h, 6h, 8h, 24h, 48h, 72h and 96h.
Carry out the bacterial strain of this type sign:
·Taurus01
·Taurus03
·Taurus04
·Taurus10
In the SSF technique of corn cob slurry (the whole slurries described in part c) utilizing 7.5%WIS, equally clearly the visible wood sugar also showing the improvement of bacterial strain of the present invention in this type of technique consumes ability (table 4).In addition, also as seen in the sign of other types, the by product of Xylitol is formed and reduces (Fig. 6).
Table 4. the cellulose degrading enzyme of the corn cob slurry and 5FPU/gWIS that utilize 7.5%WIS SSF experiment in wood sugar consumption and Xylitol formed.Cultivate in duplicate and follow the trail of 96h.
It is also use the material birch of another kind of type to carry out that SSF characterizes, and birch is the lignocellulosic material that a kind of Xylose Content is high.By birchwood crumb diluted acid (about 1%SO
2, 190 ° of C, and the residence time be 5min, carry out in SEKABE-technology company) pre-treatment.Pretreatment slurry is operated under 7.5%WIS, and under this material context, also shows the wood sugar consumption (Fig. 6) of improvement.
e) the batch feed fermentation of corn cob
Because wood sugar consumption carries out under glucose suppresses (also see Fig. 4, after glucose exhausts, wood sugar consumes initial), the another kind of setting of design is used for fermentation hydrolysis product.Corn cob liquid (described in part c) is used in fermentation, and Starting glucose charging after glucose exhausts.
Cell is produced in the mode identical with SSF technique as described above.Cell is loaded 800ml by corn cob liquid and the enrichment medium that forms as the nutrition that is used for SSF technique.At the end of batch feed, the concentration of corn cob liquid, nutrition and cell corresponds to similar SSF technique.In this experiment, corn cob liquid is diluted to the WIS content corresponding to 7.5%.Add cell and start glucose solution after 2h with in 5.53ml/h feed-in reactor during the rest part cultivated.Metabolite sample is obtained after 0h, 2h, 4h, 6h, 8h, 24h, 48h and 72h.
Carry out the bacterial strain of this type sign:
·Taurus04
This set is extremely successful, and nearly all wood sugar is all consumed (Fig. 7) and is formed more than 25g ethanol during 72h and not yet stop at this moment producing.
f) on a lbmc agar plate inhibitor tolerance test
Make the bacterial strain adaptation of the second section of the orthogenesis scheme from use cultured continuously to increase their inhibitor tolerance.Therefore, plate analysis (plateassay) is used to assess the inhibitor tolerance of these bacterial strains.
With sterile distilled water dilution from freezing sample cell sample with obtain be 1 OD.Use distilled water to carry out ten times of dilution series from this diluent, and each diluent 10 μ l dropped in YPD(rich medium, yeast extract, peptone, glucose that one has the/inhibitor mixture of 12 kinds of described in example 1 compounds above) on agar plate.These plates are cultivated at least 2 days, to allow Growth of Cells and clearly visible under 30 ° of C.
Carry out the bacterial strain of this type sign:
·Taurus03、Taurus04、Taurus05、Taurus06
As seen in Figure 8, compared with parent strain Taurus03, the bacterial strain of adaptation shows better growth in rarer sample (the 2nd row).
g) anaerobically fermenting of bagasse hydrolysate in shake-flask culture
Also in the fermentation of bagasse hydrolysate, study the performance in actual industrial substrate.
For preculture, 100 μ l are added to from the freezing cell of stock culture and has in the 100ml minimum medium of 20g/l glucose and 20g/l wood sugar.Culture is cultivated in aerobic mode under 30 ° of C and to supplement after 24h in some bagasse hydrolysates to shaking flask and to make it cultivate 6,5h again.Pass through centrifugal collecting cell.For fermentation test, the bagasse hydrolysate (20%, 50% and 75%) of different concns is supplemented with yeast extract (10g/l) and pH value is set to 6.0.Add the cell collected from pre-culture, obtain the approximate initial cell density of often liter of 3g dry weight.Use 50ml substratum in 100ml shaking flask, under 30 ° of C and 180rpm, carry out fermentation test, and by using the vent plug of filling with glycerine to obtain anaerobic condition.During this technique, follow the trail of the OD of 20% hydrolysate fermentation and measure under 650nm.
Carry out the bacterial strain of this type sign:
·Taurus03、Taurus04、Taurus07、Taurus10
Watch 5, watch 6 and watch 7 and Fig. 9 show that s-generation wood sugar bacterial strain Taurus04 with Taurus07 grows faster and provide higher ethanol production, higher xylose rate and less Xylitol to be formed compared with their parent strain Taurus03.In addition, the Taurus10 obtained from parent strain Taurus09 is also compared.Although xylose rate is large, the output of by product Xylitol all remains on low-level in all cases.When hydrolysate is diluted to 20%, xylose rate is all higher than 99% in all cases, see Fig. 9.
The ethanol production of table 5. in the anaerobism shake-flask culture utilizing 20% bagasse hydrolysate, wood sugar consumption and final alcohol concn.Carry out single cultivation.
The ethanol production of table 6. in the anaerobism shake-flask culture utilizing 50% bagasse hydrolysate, wood sugar consumption and final alcohol concn.Carry out single cultivation.
The ethanol production of table 7. in the anaerobism shake-flask culture utilizing 75% bagasse hydrolysate, wood sugar consumption and final alcohol concn.Carry out single cultivation.
h) SSF(synchronous glycosylation and the fermentation of wheat stalk slurry is utilized) experiment
The another kind of typical process that ethanol produces is SSF technique.In this technique, lytic enzyme adds together with yeast, to realize the fermentation of cellulosic decomposition in pretreated slurries and monomer sugar simultaneously.In the process, substrate and processing step is used to simulate a kind of industrial situation as much as possible.
Cell proliferation is carried out in the batch feed culture of ventilation.Preculture for breeding is carried out in the 50ml minimum medium (pH6.0) with 20g/l glucose and 20g/l wood sugar in 150ml shaking flask.With sub-fraction (cell that 100 μ l are freezing) this preculture initial and in a gyrate shaker, under 30 ° of C, carry out best 24h(18h-36h).In this batch, substratum (500ml) comprises 50g/l molasses and 23.5g/l (NH
4)
2sO
4, 3.0g/lKH
2pO
4, 2.25g/lMgSO
47H
2o and 99 μ g/l vitamin Hs, 360ppm tie up his hops (the hops extract for anti-bacteria) and 1.2ml/l defoamer (based on silicon, Nuo Pu section ENA-309, Nuo Puke Zhi Ye technology company).Add whole pre-culture with initial cultivation (inspiring confidence in gloomy bio-reactor at Lai Bu, Yi Fusen company), this cultivation carries out under the following conditions: with 1vvm ventilation, with 3MNaOH, pH value is adjusted to 5.0, and stirring velocity is 700.Carry out this batch until sugar runs out (about 8h-10h) and then initial charge.Be about in the SSF carried out, use the charging (pH value is adjusted to 5, is diluted to corresponding to 5%WIS) comprising the hydrolysate of material, and comprising molasses, thus producing total hexose sugar concentration of 75g/l.When the batch feed stage is initial, agitator speed is increased to 1000rpm and ventilation be increased in final volume body 1.0vvm.Charging is with 0.1h
-1constant rate of speed increase and carry out 20h, thus produce the final volume of 1.5l.By centrifugal (under 1800 × g 8min) collecting cell and settling flux in aseptic 0.9%NaCl.
Utilize 5%WIS and be supplemented with 0.5g/l (NH
4)
2hPO
4, the 125ppm slurry (pH5.0 regulates with 3MNaOH) of tieing up the pretreated ligno-cellulosic materials of his hops and 0.4ml/l defoamer (identical with breeding) carries out SSF part (total substratum 1.5kg).Add the cell of settling flux in 0.9%NaCl, obtain the initial cell concentration of often liter of 3g dry weight, and add enzyme (CellicC-tec2, Novi believes), obtain 10FPU/gWIS with this technique initial.Controlled, at 35 ° of C, with 3MNaOH, pH value to be adjusted to 5.0 by fermentation container (not containing plate washer, Lai Bu inspires confidence in gloomy), agitator speed be about 300rpm-400rpm, and the outlet that all entrances are all closed and pass condenser is opened.At least obtain the sample being used for metabolite analysis in the following time: 0h, 2h, 4h, 6h, 8h, 24h, 48h, 72h, 96h and 137h.
Carry out the bacterial strain of this type sign:
·Taurus07
In the SSF technique of wheat stalk slurry utilizing 5%WIS, equally clearly the visible wood sugar also showing the improvement of bacterial strain of the present invention in this type of technique consumes ability.In addition, also as seen in the sign of other types, the by product of Xylitol is formed and reduces, see Figure 10.
The solubility wood sugar perrcentage depletion of table 8. in the SSF experiment of the wheat stalk slurry of 5%WIS and 10FPU/gWIS cellulose degrading enzyme and Xylitol are formed.
i) in an Industrial demonstration scale fermentation equipment, use assessment and the fermentation of Wine brewing yeast strain Taurus04
yeast culture
Yeast culture Taurus04 breeds in three steps, and culture volume increases.In third step, at 10m
3culture tank in breed yeast.Use and breed with fed-batch mode based on the substratum of molasses.This breeding provides the yeast biomass of the amount being enough to be used in hereafter described SSF technique and hydrolysate fermentation.
During in batches, the output of propagation steps is 0.25g/g and is 0.36g/g during batch feed.Between whole nursery stage, alcohol concn is 0g/l, and by the viewpoint of fermenting, this is best.If find ethanol in the medium, so the output of yeast cell reduces, and this is undesirable.Therefore, yeast Taurus04 eugonic in large scale.
the pre-treatment of corn cob
The corn cob resistates of starting material for being provided by Hungary (Hungary).The dry weight of this material is about 85%.With dilute sulphuric acid as catalyzer with these starting material of processing parameter pre-treatment shown in hereafter, to provide slurry.
In two experiments, add when starting and be less than 200kg filtrate/slurry.Also water and yeast slurry are added in each fermentation container at first.Also add some other chemical, these chemical are shown in hereinafter together with processing parameter.
| Temperature, ° C | 35 |
| PH value | 5,5 |
| NH 3 | 3 |
| H 3PO 4 | 1 |
| Froth suppressor, L | 2 |
| Tie up his hops S, L | 0,5 |
| Filtrate/slurry original bulk, kg | 200 |
In Section 1 experiment (hydrolysate fermentation), filter slurry and only use liquid portion.At this experimental session, add whole 2840 liters of filtrates and about 1144 liters of extra liquid.Continue to add filtrate during the initial 24h of this experiment.
Initial interpolation correspond to 5.3FPU/gWIS(water insoluble solid) enzyme, to be hydrolyzed oligomeric carbohydrates.Yeast concn calculates with dry weight for about 5g/l(, DS).
result:observe wood sugar completely consumed.The xylose of 10% is lactic acid and Xylitol, and all the other wood sugars of 90% are converted into ethanol.The ultimate density of ethanol is 10,9g/l, see Figure 11 a.
Inhibitor HMF and furfural are by yeast detoxification, and the concentration of these inhibitor is 0g/l in whole experiment.Also observe, along with the feeding of slurry during initial 24h, glucose and wood sugar are all consumed with identical speed, and after this produced ethanol necessarily derives from wood sugar, because hexose is consumed.
In Section 2 experiment (SSF technique), add whole slurry.Add 2844kg slurry and 1092 liters of liquid.In addition, the enzyme of the enzymic activity corresponding to 15FPU/gWIS is also added.Yeast concn is 5.0g/l(TS).
result:in Section 2 experiment, in the concentration of whole experimental session glucose and seminose close to 0g/l, the concentration of lactic acid is also like this.Yeast stably consumes wood sugar and reaches about 100h.The effective fermentation of hexose and wood sugar produces the final ethanol concentration of 39.4g/l, and this corresponds to the transformation efficiency that wood sugar forms about 40% of ethanol, see Figure 11 b.
j) in the tolerance utilizing wood sugar growing period bacterial strain Taurus01, Taurus03, Taurus04, Taurus07, Taurus09 and Taurus10 to acetate and HMF
Experimental detail:
Preparation 50g/l or 100g/l and pH value is the xylose media of 5.0, this substratum comprises acetate and the HMF of 10g/l yeast extract, 100mM phthalic acid potassium and different levels.Add in the acetate of sodium acetate form, produce 0g/l, 4g/l, 7g/l or 10g/l(zero, low, in and high) acetic acid level.Add 0g/l, 2g/l or 5g/l(zero, low and high) HMF.All compounds are all dissolved in a certain amount of water, adjust ph, are then diluted to final volume and by filter sterilizing solution.To aseptic culture medium pH value determination and this pH value between 4.94-5.15.All combinations all produce 24 kinds of dissimilar substratum.Substratum is labeled as the 1-12 of 50g/l wood sugar and the 13-24 for 100g/l wood sugar.These acetate levels are divided into three groups, for zero, low, in and high level be numbered 1-3,4-6,7-9,10-12(respectively and make corresponding numbering for high wood sugar).In each group in three groups, HMF level is increased to low and high (such as 1,2,3) from zero.The above is found in Figure 10.But this figure does not show situation when there is not HMF or acetate concentration.In addition, the figure of test 11 and 12 does not show in Fig. 10, because excessive for environmental toxicity bacterial strain.
YPDX substratum is used to carry out inoculum cultivation and grow about 24h.By centrifugal collecting cell and settling flux in aseptic mQ water.Measure biomass concentration, and by preparing the cell suspending liquid of often liter of about 3g dry weight with aseptic mQ water dilution.
At biological grid (BioScreen) (growth curve company of Finland (GrowthCurvesOY, Finland)) under 30 ° of C under continuous oscillation, carry out fermentation lasts 72h in triplicate with 170 μ l substratum and 30 μ l cell suspending liquids, produce the starting cell concentration of often liter of 0.46g dry weight.The condition of this biological grid is half aerobic.At the end of cultivation, the substratum from triplicate culture is mixed and filters and analyzes (at Bole HPX87H post), to measure tunning, HMF, acetate and wood sugar for HPLC.
Result:
Measure wood sugar consumption, ethanol production and growth characteristics, and bacterial strain Taurus07 have higher HMF level and higher acetate level have more challenging condition under show better performance, see Figure 12.High wood sugar level applies extra-stress to the problematic cell of tool under higher HMF level.
k) fermentation to C5 material of Taurus04 bacterial strain is used
Be 50g/L-60g/L by C5 liquid diluting to xylose concentration.Dilute according to the xylose concentration in this liquid.The pH value of liquid is adjusted to 5.5(KOH).Distribute the liquid in 125mL Erlenmeyer flask (Erlenmeyerflask), each flask 60mL liquid.Add the antibiotic (penicillin G (penicillinG) or virginiamycin (virginiamycin)) of 5ppm to prevent bacterial contamination.Add 0.06g/L(1mM) urea and the yeast extract (nutrition is provided) of 0.5g/L.With often liter of 0.5g dry-pitching yeast.Under 32 ° of C, (in the shaking bath of 125rpm) cultivates flask.
Wood sugar is consumed effectively, and after 48h, residue is less than 20% wood sugar.During the major part of fermentation, produce ethanol, after 64h, reach maximum horizontal 10.1g/l.This generates often consume one gram of sugared 0.34g ethanol ethanol production (to 64h).Only during the wood sugar stage, the ethanol production of (namely from time 17h to 64h) is often consume one gram of wood sugar 0.30g ethanol, see Figure 13 a.
l) fermentation of the C6 solid through hydrolysis of Taurus04 is used
About the initial adaptation of cell in inoculum, by 30ml through the centrifugal and C6 hydrolysate of sterilising filtration to add in culture (with identical mode of fermenting about C5,5h cultivates the time).About C5 fermentation, use 10MKOH adjust ph, and urea and yeast extract are dissolved in the adjusted hydrolysate of pH value, with dilution water hydrolysis products as few as possible (remaining particle is not taken away after hydrolysis).Starting cell concentration in main cultivation is often liter of 0.5g dry weight.
Sugar is consumed effectively.After being less than 24h, all glucose all runs out and wood sugar again more than 85% after 48h is consumed.Ethanol mainly produces during initial 48h, reaches maximum horizontal 22.8g/l after 46h.This generates often consume one gram of sugared 0.39g ethanol ethanol production (to 46h).Only during the wood sugar stage, the ethanol production of (namely from time 22h to 46h) is often consume one gram of wood sugar 0.34g ethanol, see Figure 13 b.
m) the C5 material of Taurus04 bacterial strain and the synchronous glycosylation of C6 solid and fermentation is used
Cell proliferation is carried out with batch feed form of cultivating in the YP substratum with 20g/l glucose and 20g/l wood sugar.With 5MKOH, the charging be made up of C5 liquid is adjusted to pH5.0, and supplements 40g/l sucrose.By centrifugal collecting cell and settling flux in water.
With the substratum that the final quantity of the slurry with 15% total C6 solid is tested for the preparation of SSF for 1500g.Because C6 material comprises 30% solid and 70% moisture, so 750gC6 material is mixed with 750gC5 part and with 10MKOH, pH value is adjusted to 5.5.Because the level of wood sugar and acetic acid is too low and can not represent felicity condition in material, so by 64.5g wood sugar and 3g acetic acid in slurry, obtain the total concn of about 57g/l and 4g/l respectively.Add the defoamer (polyoxyethylene glycol) of the urea of 0.06g/l, the penicillin of 5ppm and 0.2ml/l.The condition that SSF cultivates in Lai Bu inspires confidence in gloomy fermentation container (Yi Fusen company) is temperature 32 ° of C, and control 5.5 with 5MNaOH by pH value, agitator speed is 600rpm, and confining gas entrance.Initial cultivation is carried out with the CellicCtec2 enzyme (Novi's letter) obtaining often liter of 1g dry weight and every gram of Mierocrystalline cellulose 50mg crude extract by adding cell.Follow the trail of cultivate 120h by the sampling (centrifugal under 14500rpm, 2min, then filters as described above and analyze) of extracellular medium.
As shown in Figure 14, remain 20g/l wood sugar after the 120h of technique, this corresponds to 70% wood sugar dissolved and is consumed by yeast.When fermentation ends, Xylitol concentration is 9g/l, and therefore some wood sugars are unconverted is ethanol.Due to cellulolytic enzyme act on the initial 6h of technique during glucose slightly increase, but do not find that glucose is accumulated in the later stage of technique.This fact shows yeast consumption of glucose just well, and enzymically hydrolyse may be a limiting factor.Final alcohol concn is 38.1g/l.According to the original negative carrying capacity of 15% total solids, in 1.5l slurry, there is 228.75g total reducing sugar available potentially.Therefore, in total operational sugar, the ethanol produced corresponds to the output of 48% of theoretical yield, Figure 14.
Claims (8)
1. there is a Wine brewing yeast strain for the ethanol production of improvement, the xylose rate of improvement and the Xylitol output of reduction, the Taurus03 of wherein said bacterial strain to be preserving number be CBS128138.
2. there is the Wine brewing yeast strain of inhibitor tolerance for the ethanol production of improvement, the xylose rate of improvement, the Xylitol output of reduction and increase, the Taurus04 of wherein said bacterial strain to be preserving number be CBS128139.
3. there is the Wine brewing yeast strain of inhibitor tolerance for the ethanol production of improvement, the xylose rate of improvement, the Xylitol output of reduction and increase, the Taurus07 of wherein said bacterial strain to be preserving number be CBS128140.
4. the bacterial strain according to any one of claim 1-3 is used for lignocellulose raw material to ferment for the purposes of ethanol.
5. the bacterial strain according to any one of claim 1-3 is used for corn cob hydrolysate and bagasse hydrolysate to ferment for the purposes of ethanol.
6. purposes according to claim 5, the concentration of wherein said bagasse hydrolysate is 20%, 50% or 75%.
7. the bacterial strain according to any one of claim 1-3 is for the purposes in synchronous glycosylation and fermentation (SSF) technique.
8. purposes according to claim 7, for the fermentation of corn cob slurry, birch slurry and wheat stalk slurry.
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| WO2014193344A1 (en) * | 2013-05-28 | 2014-12-04 | Poet Research, Inc. | System for management of yeast to facilitate the production of ethanol |
| WO2015057148A1 (en) | 2013-10-16 | 2015-04-23 | Scandinavian Technology Group Ab | Saccharomyces cerevisae strains |
| CN103627644B (en) * | 2013-10-26 | 2015-09-30 | 三峡大学 | A kind of yeast saccharomyces cerevisiae dissociant and application thereof |
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| US11639506B2 (en) * | 2015-05-15 | 2023-05-02 | Wisconsin Alumni Research Foundation | Recombinant yeast having increased tolerance to ionic liquids and methods of use |
| CN106906152B (en) * | 2017-05-04 | 2020-06-19 | 天津大学 | Saccharomyces cerevisiae and application thereof |
| CN111100801B (en) * | 2019-09-09 | 2024-02-23 | 四川农业大学 | Furfural inhibitor tolerant yeast engineering strain, construction method and application thereof |
| CN112877273B (en) * | 2020-12-09 | 2021-08-17 | 山东太阳纸业股份有限公司 | Yeast for decomposing heterosaccharide such as galactose, mannose and the like |
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